An increasing number of proteins are known to form amyloid fibrils in vivo, and the formation of these fibrils is implicated in several diseases (e.g. Alzheimer's, Parkinson's). One of these proteins, human -2-microglobulin (2m), can form amyloid fibrils in the presence of Cu(II), and these fibrils are presumed to be the main pathogenic process underlying dialysis- related amyloidosis (DRA). Like other amyloid systems, 2m fibril formation proceeds by partial protein unfolding, subsequent oligomerization, and eventual elongation to form mature fibrils. While many aspects of general amyloid formation are understood, molecular-level information is lacking for the early stages of almost all amyloid forming reactions; however, this information is critical for the rational development of therapeutics against amyloid diseases like DRA. We intend to obtain amino acid-level information about the unfolding and oligomerization of 2m that precedes its fibril formation. To do so, we will develop, optimize, and apply three mass spectrometry (MS)-based methods with the necessary temporal and spatial resolution. (1) A new metal-catalyzed oxidation (MCO) induced deuterium labeling strategy will be investigated to complement our existing MCO/MS method. The proposed MCO-induced deuterium labeling strategy will provide Cu-2m binding data that are more easily interpreted, contain more information about all the Cu-bound amino acids, and are less prone to false positives. (2) Covalent labeling with MS detection will be used to study changes in 2m structure upon Cu(II) binding, unfolding, and oligomerization. The covalent labeling reactions will identify changes to the solvent accessibility of different amino acids in 2m as this protein progresses from monomer to oligomers. (3) Electrospray ionization (ESI) with top-down sequencing will be explored as a means to separate and characterize protein oligomers formed by 2m. The differential charging that occurs during ESI of protein oligomers will be used to rapidly separate protein oligomers, and top-down sequencing will be used to identify the solvent accessible amino acid residues that are labeled in individual oligomers. Our preliminary data on 2m fibril formation support a model in which Cu(II) is necessary to organize the dimer and an initial tetramer but is released upon formation of a second tetramer and the hexamer. We will use these three MS-based methods to study this model and identify the structural changes associated with the formation of each oligomeric state.